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New technology for correcting disease-causing mutations

Staining of RAD51 (bright cyan dots) in fertilized 1-cell mouse embryos indicates CRISPR-induced repair of DNA breaks.Credits: Massachusetts Institute of Technology

Gene editing, or deliberately altering the DNA sequence of a gene, is a powerful tool for studying how mutations cause disease and for therapeutically altering an individual’s DNA. A new method of gene editing that can be used for both purposes is now being developed by a team at the Massachusetts Institute of Technology (MIT), led by James W. (1963) in Brain and Cognitive Science and Professor Patricia T. Poitras.

“This technological advance can accelerate the creation of animal disease models and, importantly, open up entirely new methodologies for correcting disease-causing mutations,” said Harvard University and MIT. Feng, who is also a member of the Broad Institute, said: Deputy Director of the McGovern Institute for Brain Research at MIT.New findings published online in the journal cell.

Genetic model of disease

The main goal of the Feng lab is to accurately define what goes wrong with neurodevelopmental and neuropsychiatric disorders. It is by manipulating animal models that carry gene mutations that cause these disorders in humans. A new model can be generated by injecting a gene editing tool into an embryo and then injecting a piece of DNA with the required mutation.

In one such method, the gene editing tool CRISPR is programmed to cleave the target gene, which activates the natural DNA mechanism that “repairs” the broken gene in the injected template DNA. Will be done. Genetically engineered cells are then used to generate offspring that can pass on genetic changes to the next generation, creating a stable genetic lineage that is tested for disease and treatment.

CRISPR has accelerated the process of creating such disease models, but the process can still take months or years. The reason for the inefficiency is that many processed cells undergo no changes in the desired DNA sequence, and changes occur in only one of the two gene copies (most often). gene, Each cell contains two versions, one from the father and one from the mother).

To increase the efficiency of the gene editing process, Feng’s research team initially added a DNA repair protein called RAD51 to the standard mixture of CRISPR gene editing tools to give cells (in this case fertilized mouse eggs, or 1-cell embryos) Will undergo the desired genetic changes.

As a test case, they measured the rate at which mutations could be inserted (“knocked in”) into the autism-related Chd2 gene. The overall proportion of correctly edited embryos did not change, but surprisingly, the proportion of desirable gene edits on both chromosomes was significantly higher. Tests with different genes gave the same unexpected results.

“Editing both chromosomes at the same time is usually very rare,” explains postdoctoral fellow Jonathan Wilde. “The high edit rates seen with RAD51 were truly amazing. What started as a simple attempt to create a mutant Chd2 mouse is much larger with a focus on RAD51 and its application to genome editing. It quickly turned into a project, “said co-author Wilde. A cell paper with researcher Tomomi Aida.

Molecular copier

Feng’s research team then set out to understand how RAD51 enhances gene editing. They hypothesized that RAD51 was involved in a process called interhomolog repair (IHR). In this process, DNA breaks in one chromosome are repaired using a second copy of the chromosome from the other parent as a template.

To test this, they injected RAD51 and CRISPR into mouse embryos, leaving template DNA. They programmed CRISPR to cleave only one gene sequence on the chromosome and tested whether it was repaired to match the sequence on the uncut chromosome. This experiment required the use of mice with different maternal and paternal chromosomal sequences.

They found that control embryos injected with CRISPR alone showed little IHR repair. However, the addition of RAD51 significantly increased the number of embryos in which the CRISPR target gene was edited to match the uncleaved chromosome.

“Previous studies of IHR have shown that IHR is very inefficient in most cells,” says Wilde. “Our finding that it develops more easily in germ cells and can be enhanced by RAD51 is a safer and more efficient gene therapy design to better understand what embryos allow for this type of DNA repair. Suggests that it helps. “

A new way to fix disease-causing mutations

A standard gene therapy strategy that relies on injecting a modified portion of DNA that acts as a template for repairing mutations involves a process called homologous recombination repair (HDR).

“HDR-based strategies remain inefficient and carry the risk of unwanted integration of donor DNA throughout the genome,” Feng explains. “IHR has the potential to overcome these problems because it relies on the natural cellular pathway and the patient’s own normal chromosomes to correct harmful mutations.”

Feng’s team further identified DNA repair-related proteins that could stimulate IHR. Some of them not only promote high levels of IHR, but also reduce errors in the DNA repair process. Additional experiments that allowed the team to study the genomic function of IHR events provided deeper insight into the mechanism of IHR and suggested ways to use this technique to make gene therapy safer.

“There is still much to learn about this new application of IHR, but our findings are the basis for a new gene therapy approach that may help solve some of the major problems of our current approach.” Aida says.


New approaches to gene therapy can correct mutations that cause diseases in genes


For more information:
Efficient embryo homozygous gene conversion by interhomolog repair enhanced by Jonathan J. Wilde et al, RAD51, cell (2021). DOI: 10.1016 / j.cell.2021.04.035

Journal information:
cell

This article is from MIT Newsweb.mit.edu/newsoffice/) Is a popular site that covers news about MIT’s research, innovation and education.

Quote: A new method for correcting disease-causing mutations (June 10, 2021) was posted on June 10, 2021 at https://medicalxpress.com/news/2021-06-technique-disease- Obtained from causing-mutations.html.

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New technology for correcting disease-causing mutations

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